Tissue regeneration promoting sheet

Abstract

A tissue regeneration promoting sheet (10) is provided with a cell holding sheet (12) which is a non-woven fabric that contains fibers mainly composed of polycaprolactone and is capable of allowing a cell suspension containing stem cells to permeate therethrough. The fiber diameter of the fibers constituting the non-woven fabric may be in the range of 300 nm to 2 [mu] m. The weight per unit area of the non-woven fabric may be in the range of 1 g / m2 to 3 g / m2. The thickness of the non-woven fabric may be 300 [mu] m or more. The tissue regeneration promoting sheet (10) may also be provided with a reinforcing sheet (14) which is laminated on the nonwoven fabric and into which the cell suspension can permeate.

Description

Technical Field

[0001] The present invention relates to a tissue regeneration promoting sheet. Background Art

[0002] In recent years, in the field of regenerative medicine, a tissue regeneration promoting material is being developed that can hold cells inside and promote tissue regeneration by being embedded in the body. For example, Patent Document 1 discloses a sheet-like tissue regeneration promoting material, on the surface of which at least one of the group consisting of white blood cells and platelets exists. In addition, Patent Document 2 discloses a liquid tissue regeneration promoting material obtained by mixing a powder of a polymer material with cells.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent document 1: Japanese Patent No. 4847129;

[0006] Patent document 2: Japanese Patent Application Publication No. 2015-112262. Summary of the invention

[0007] Technical problem to be solved by the invention

[0008] However, in the tissue regeneration promoting materials disclosed in Patent Documents 1 and 2, since porous bodies and polymer powders are used, there is a problem that it is difficult to stably maintain mesenchymal stem cells with high tissue regeneration ability in cells. Such a problem is not limited to the case where mesenchymal stem cells are maintained by the tissue regeneration promoting material, but also exists in the case where other stem cells are maintained.

[0009] The present invention has been made based on such a background, and an object of the present invention is to provide a tissue regeneration promoting sheet capable of stably retaining stem cells.

[0010] Solutions to technical problems

[0011] In order to achieve the above-mentioned object, the tissue regeneration promoting sheet nonwoven fabric of the present invention is:

[0012] The nonwoven fabric contains fibers containing polycaprolactone as a main component and is capable of permeating a cell suspension containing stem cells.

[0013] The fiber diameter of the fibers constituting the nonwoven fabric may be in the range of 300 nm to 2 μm.

[0014] The unit area weight (weight per unit area) of the nonwoven fabric can be 1 g / m 2 ~3g / m 2 within the range.

[0015] The nonwoven fabric may have a thickness of 300 μm or more.

[0016] The tissue regeneration promoting sheet may further include a reinforcing sheet which is laminated on the nonwoven fabric and can allow the cell suspension to permeate.

[0017] The reinforcing sheet may be another nonwoven fabric composed of fibers coarser than the fibers constituting the nonwoven fabric.

[0018] The nonwoven fabric may include a first nonwoven fabric and a second nonwoven fabric, wherein the second nonwoven fabric is stacked on the first nonwoven fabric and is less likely to deform than the first nonwoven fabric.

[0019] Effects of the Invention

[0020] According to the present invention, a tissue regeneration promoting sheet capable of stably retaining stem cells can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a perspective view showing the structure of a tissue regeneration promoting sheet according to an embodiment of the present invention.

[0022] Figure 2 This is a front view showing a state of manufacturing the tissue regeneration promoting sheet according to the embodiment of the present invention.

[0023] Figure 3 It is a perspective view showing the structure of a tissue regeneration promoting sheet according to a modified example of the present invention.

[0024] Figure 4 It is a perspective view showing the structure of a tissue regeneration promoting sheet according to another modified example of the present invention.

[0025] Figure 5 This is a graph showing the capture rate of mesenchymal stem cells by each sample in Example 1.

[0026] Figure 6 This is a graph showing the capture rate of mesenchymal stem cells by each sample in Example 2.

[0027] Figure 7 The figure shows the CT image of the rat cervical spine in Example 3.

[0028] Figure 8 The figure shows the immunostaining image at 6 weeks after the bone transplantation in Example 3. DETAILED DESCRIPTION

[0029] Hereinafter, a tissue regeneration promoting sheet according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings. In each of the drawings, the same or equivalent parts are denoted by the same reference numerals.

[0030] The tissue regeneration promoting sheet material involved in the embodiment is a sheet material that promotes tissue regeneration at the damaged part by being buried in the damaged part in the body. The tissue regeneration promoting sheet material involved in the embodiment has a non-woven fabric composed of fine-diameter fibers, such as microfibers, to which stem cells are attached. The non-woven fabric is a sheet material that is wound without weaving fine-diameter fibers. The non-woven fabric can be deformed freely and is therefore suitable for embedding or pasting according to the shape of the damaged part. The tissue regeneration promoting sheet material can be pasted or buried on any internal organs, such as the heart, blood vessels, lungs, trachea, esophagus, stomach, small intestine, large intestine, liver, kidneys, bladder, bones, muscles, and skin. The tissue regeneration promoting sheet material can function as a scaffold that promotes the proliferation of specific cells in the body.

[0031] like Figure 1 As shown, the tissue regeneration promoting sheet 10 includes a cell holding sheet 12 for holding stem cells and a pair of reinforcing sheets 14 that are joined to and reinforce the cell holding sheet 12 in a manner of sandwiching the cell holding sheet 12 from above and below. The cell holding sheet 12 is sandwiched and reinforced by a pair of reinforcing sheets 14 because the nonwoven fabric constituting the cell holding sheet 12 is easily rounded when wet due to the raw material properties described later. The cell holding sheet 12 and the reinforcing sheet 14 can be joined to each other by an adhesive or heat fusion, or can be joined by entanglement of fibers when the nonwoven fabric is manufactured.

[0032] The number of cell-retaining sheets 12 disposed between a pair of reinforcing sheets 14 is arbitrary within the range capable of filtering a cell suspension containing stem cells, but in order to prevent the cell suspension from leaking from the support frame described later when filtering the cell suspension, it is preferred to stack two or more sheets. A cell suspension is a liquid that allows cells to float in a liquid that can be administered into the body. When stacking a plurality of cell-retaining sheets 12, they can be joined to each other in the same manner as when stacking the cell-retaining sheets 12 and the reinforcing sheets 14. In addition, in Figure 1 In the figure, the thickness direction of the tissue regeneration promoting sheet 10 is exaggerated for easy understanding. The thickness of the tissue regeneration promoting sheet 10 is set to a degree that allows filtering of a cell suspension.

[0033] The cell retaining sheet 12 is a non-woven fabric containing fibers whose main component is polycaprolactone. The inventors conducted in-depth research and found that in the non-woven fabric containing fibers whose main component is polycaprolactone, stem cells can be efficiently captured when filtering a cell suspension containing stem cells, and the activity of attached stem cells can be promoted. Polycaprolactone is a semi-crystalline and biodegradable thermoplastic polyester, and is also suitable for the manufacture of non-woven fabrics. Polycaprolactone is, for example, poly-ε-caprolactone. The molecular weight of polycaprolactone can be a molecular weight suitable for injection molding, for example, in the range of 70,000 to 100,000.

[0034] The fiber with polycaprolactone as the main component may be formed only of polycaprolactone or may contain other substances. As other substances, for example, agents that promote tissue regeneration and other biodegradable polymers may be used. Agents that promote tissue regeneration are, for example, hydroxyapatite (calcium hydroxide phosphate) and tricalcium phosphate. Other biodegradable polymers are, for example, polylactic acid, polyglycolic acid, polydioxanone and copolymers thereof.

[0035] The thinner the fiber diameter of the fiber constituting the cell holding sheet 12, the higher the capture rate of stem cells. However, the cell holding sheet 12 tends to become round when absorbing water. Considering the capture rate of stem cells and the operability, the fiber diameter of the fiber constituting the cell holding sheet 12 is, for example, in the range of 100 nm to 3 μm, preferably in the range of 500 nm to 1 μm.

[0036] The greater the weight per unit area of ​​the cell holding sheet 12, the greater the amount of attached stem cells, but the filtration of the cell suspension becomes increasingly difficult. Considering the amount of attached stem cells and the ease of manufacturing, the weight per unit area of ​​the cell holding sheet 12 is, for example, 1 g / m 2 ~3g / m 2 In the range of 1.5 g / m 2 ~2.5g / m 2 In the range of 2 g / m 2 .

[0037] Considering the operability of the forceps, the thickness of the cell holding sheet 12 is preferably 300 μm or more, and more preferably 500 μm or more, for example. However, it should be noted that the thicker the cell holding sheet 12 is, the more difficult it is to filter the cell suspension.

[0038] The stem cells attached to the cell holding sheet 12 are preferably mesenchymal stem cells (MSC) or cells differentiated from mesenchymal stem cells. Mesenchymal stem cells are somatic stem cells that can differentiate into cells belonging to the mesenchyme, and can be derived from fat, periosteum, synovium, spongy bone, bone marrow, amniotic membrane, umbilical cord blood, or placenta. In addition, the stem cells attached to the non-woven fabric can be, for example, ES (Embryonic Stem) cells and iPS (induced Pluripotent Stem) cells. It is also possible to attach multiple stem cells to the non-woven fabric together.

[0039] The stem cells attached to the cell holding sheet 12 may be stem cells collected from a patient. In order to culture stem cells in vitro, for example, a culture medium containing fetal bovine serum (FBS) or human serum (preferably human serum from a patient), or a serum-free culture medium may be used. The stem cells cultured in the culture medium may be directly attached to the nonwoven fabric, or the stem cells may be differentiated and expressed in specific cells, such as osteoblasts, and then attached to the nonwoven fabric. In order to express specific cells, for example, a differentiation inducing factor may be added to the culture medium of the stem cells.

[0040] Furthermore, stem cells do not necessarily need to be attached to the cell retaining sheet 12 in advance. After the tissue regeneration promoting sheet 10 is placed at the damaged site in the body, a cell suspension containing stem cells may be administered into the vascular system to allow the cell retaining sheet 12 to capture the stem cells in the body.

[0041] The reinforcing sheet 14 comprises fibers with a biocompatible polymer as the main component, and is a nonwoven fabric that allows a cell suspension to pass through. The reinforcing sheet 14 is joined to the cell holding sheet 12 in order to reinforce the cell holding sheet 12 that is easy to curl and deform. The biocompatible polymer constituting the reinforcing sheet 14 is a biocompatible polymer with strength compared to polycaprolactone, such as biodegradable polymers such as chitin, chitosan, polylactic acid, and polyglycolic acid, wherein chitin is preferred. The fiber diameter of the microfibers of the reinforcing sheet 14 is preferably thicker than the fiber diameter of the microfibers of the cell holding sheet 12. In addition, although it is not necessary to attach stem cells to the reinforcing sheet 14, stem cells can also be attached to the reinforcing sheet 14 in the process of attaching stem cells to the cell holding sheet 12.

[0042] The above is the structure of the tissue regeneration promoting sheet 10 .

[0043] Next, the specific steps of attaching stem cells to the cell holding sheet 12 according to the embodiment are described. To attach stem cells to the cell holding sheet 12, it is sufficient to contact the cell turbid solution containing stem cells for a certain period of time, but in order to suppress the reduction of the growth factor production ability during stem cell culture, it is preferably carried out according to the following steps.

[0044] First, if Figure 2 As shown, a cell holding sheet 12 and a reinforcing sheet 14 are stacked between a pair of support frames 20 constituting the filtration device. Each support frame 20 is configured to allow the cell turbidity to flow out or in, and when the cell suspension flows from one support frame 20 to the other support frame 20, the cell turbidity comes into contact with the cell holding sheet 12. Figure 2In the embodiment, although there is a gap between the two cells, the pair of support frames 20 are actually in close contact with each other in a joined state where the two cell holding sheets 12 are sandwiched between the pair of reinforcing sheets 14 .

[0045] Next, the cell turbidity containing stem cells is slowly dripped from above drop by drop, and the cell turbidity is filtered through two cell retaining sheets 12. The filtration speed is, for example, a speed of dripping 1 ml of the cell turbidity in 2 to 3 minutes. At this time, the stem cells contained in the dripped cell suspension spontaneously adhere to the fibers of the cell retaining sheet 12. The strict mechanism is not yet clear, but if the cell turbidity containing stem cells is filtered little by little through a non-woven fabric composed of fibers with polycaprolactone as the main component, the stem cells are stably attached to each fiber, and the attached stem cells are activated to promote the production of growth factors.

[0046] The above are specific steps for making stem cells adhere to the cell holding sheet 12 .

[0047] Furthermore, as long as the stem cells are attached to the cell retaining sheet 12, it can be expected that some growth factors will be produced from the stem cells, and therefore the above-mentioned steps may not necessarily be used.

[0048] As described above, the tissue regeneration promoting sheet 10 according to the embodiment includes: a cell holding sheet 12 that includes fibers having polycaprolactone as a main component and that can be permeated with a cell suspension containing stem cells; and a reinforcing sheet 14 that is laminated on the cell holding sheet 12 and that can be permeated with the cell suspension. Therefore, the stem cells can be stably held by the cell holding sheet 12, and the operability of the user can be improved.

[0049] The present invention is not limited to the above-described embodiment, and modifications as described below are possible.

[0050] (Variation Example)

[0051] In the above embodiment, two cell-retaining sheets 12 are arranged between the pair of reinforcing sheets 14, but the present invention is not limited thereto. The number of cell-retaining sheets 12 sandwiched between the pair of reinforcing sheets 14 may be one or three or more.

[0052] In the above embodiment, the reinforcing sheet 14 is a nonwoven fabric, but the present invention is not limited thereto. For example, the reinforcing sheet 14 may be a porous body that allows the cell suspension to permeate.

[0053] In the above embodiment, the reinforcing sheet 14 is clamped from above and below relative to the cell holding sheet 12, but the present invention is not limited thereto. Figure 3As shown in the figure, the tissue regeneration promoting sheet 10 may be formed by stacking a reinforcing sheet 14 on one surface of the cell holding sheet 12. In the case where the strength of the tissue regeneration promoting sheet 10 can be ensured even if the reinforcing sheet 14 is omitted, or in the case where the strength of the tissue regeneration promoting sheet 10 does not need to be ensured, the reinforcing sheet 14 may be omitted. The case where the strength of the tissue regeneration promoting sheet 10 does not need to be ensured is, for example, the case where the sheet is accommodated in a space within an implant or is kneaded and buried in a damaged part in the body.

[0054] In order to ensure the strength of the tissue regeneration promoting sheet 10 even if the reinforcing sheet 14 is omitted, for example, at least one of the fiber diameter and fiber density of the fibers constituting the cell holding sheet 12 and the thickness of the cell holding sheet 12 may be increased. Figure 4 As shown, the tissue regeneration promoting sheet 10 may be a laminate of a first cell holding sheet 12A and a second cell holding sheet 12B that is less deformable than the first cell holding sheet 12A. The second cell holding sheet 12B may be, for example, made thicker in fiber diameter or have a higher fiber density than the first cell holding sheet 12A. The first cell holding sheet 12A is an example of a first nonwoven fabric, and the second cell holding sheet 12B is an example of a second nonwoven fabric that is less deformable than the first nonwoven fabric.

[0055] The above-mentioned embodiments are illustrative, and the present invention is not limited to these. Various embodiments can be performed without departing from the scope of the invention described in the claims. The constituent elements described in the embodiments and modifications can be freely combined. In addition, inventions equivalent to the inventions described in the claims are also included in the present invention.

[0056] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

[0057] Example

[0058] (Example 1)

[0059] In Example 1, a sheet of a laminated polycaprolactone (PCL) nonwoven fabric and a chitin nonwoven fabric was prepared using the method described in the above embodiment, and the capture rate of mesenchymal stem cells of each was evaluated. Specifically, first, Figure 1 As shown in the figure, two PCL nonwoven fabrics are joined from top to bottom, sandwiching the chitosan nonwoven fabric. The fiber diameter of the PCL nonwoven fabric is 0.5 to 1 μm, and the fiber density is 2 g / m 2 The fiber diameter of chitosan nonwoven fabric is 8μm and the fiber density is 30g / m 2. Next, a cell suspension containing human mesenchymal stem cells was passed through the filter while being clamped in a filter sterilization support frame, thereby attaching the mesenchymal stem cells to the PCL nonwoven fabric, and the capture rate of the mesenchymal stem cells was evaluated. As a support frame, a Swinnex filter holder (Merckmillipore) was used. As a comparative example, the same experiment was also performed on a sheet of only two chitin nonwoven fabrics stacked. The experiments were performed on three samples respectively.

[0060] The results are shown in Figure 5 The average cell capture rate in the sheet with only the chitin nonwoven fabric laminated was 43.6%, whereas the average cell capture rate in the sheet with the PCL nonwoven fabric and the chitin nonwoven fabric laminated was 100%. From the above, it can be confirmed that the PCL nonwoven fabric allows stem cells to attach efficiently.

[0061] (Example 2)

[0062] In Example 2, a sheet of a laminated PCL nonwoven fabric and a polyester (PEs) nonwoven fabric, a sheet of a PCL nonwoven fabric alone, and a sheet of a laminated PCL nonwoven fabric with two different properties were prepared to verify operability. In addition, a cell suspension was dripped into each sample to measure the cell capture rate.

[0063] An attempt was made to place a sheet of PCL nonwoven fabric alone on the support frame. It was found that when the thickness of the PCL nonwoven fabric was less than 200 μm, the PCL nonwoven fabric alone could not be placed on the support frame. Therefore, when the thickness of the PCL nonwoven fabric was less than 200 μm, the PCL nonwoven fabric and the PEs nonwoven fabric were stacked, and when the thickness of the PCL nonwoven fabric was more than 300 μm, the sheet of PCL nonwoven fabric alone was placed on the support frame. In addition, a fiber diameter of 0.5 μm and a fiber density of 2 g / m 2 , PCL nonwoven fabric with a thickness of 100 μm and a fiber diameter of 4.9 μm and a fiber density of 10 g / m 2 The sheet was formed by laminating PCL nonwoven fabrics with a thickness of 500 μm, and the sheet was able to be set on the support frame. The other steps were the same as in Example 1. Three samples were used for each condition, but for the fiber diameter of 0.5 μm and the fiber density of 2 g / m 2 Only one sample was used for the experiment, with a thickness of 100 μm, 2 PEs layers, and 3 to 6 PCL layers.

[0064] The results are shown in Figure 6. If we focus on the data group with 2 PEs stacking numbers, 2 PCL stacking numbers, and a thickness of 200μm, it shows that the thinner the fiber diameter, the higher the cell capture rate. Specifically, when the fiber diameter is 0.5μm, the cell capture rate is 100±0.0%, while in contrast, when the fiber diameter is 2.8μm, the cell capture rate is 73.4±18.8%. In addition, if we focus on the data group with a fiber diameter of 4.9μm, it shows that the greater the thickness, the higher the cell capture rate. Specifically, when the thickness is 200μm, the cell capture rate is 74.9±9.5%, and when the thickness is 500μm, the cell capture rate is 99.4±3.6%. From the above, it can be confirmed that the thinner the fiber diameter and the greater the thickness, the higher the capture rate of mesenchymal stem cells.

[0065] (Example 3)

[0066] In Example 3, a sheet formed by laminating PCL nonwoven fabric and chitin nonwoven fabric was transplanted in combination with a bone sheet on the cervical vertebrae of three immunodeficient model rats, and bone fusion was evaluated manually after 6 weeks after transplantation. In addition, the evaluation of bone fusion was also implemented using CT (Computed Tomography) images of the cervical vertebrae of immunodeficient model rats and immunostaining images of the collected tissue sheets. The immunostaining images were obtained by photographing tissue sheets stained with a fluorescent dye (i.e., DAPI (4', 6-diamidino-2-phenylindole)) bound to DNA (Deoxyribonucleic acid) using a fluorescence microscope. In a sheet formed by laminating PCL nonwoven fabric and chitin nonwoven fabric, a sheet with human mesenchymal stem cells attached was used. As a comparative example, bone transplantation was performed only on the cervical vertebrae of five immunodeficient model rats, and bone fusion was evaluated in the same manner.

[0067] As a result, in the nonwoven fabric combination group using a sheet made of a laminated PCL nonwoven fabric and a chitin nonwoven fabric, 3 out of 3 animals underwent bone fusion, and the bone fusion rate was 100%. On the other hand, in the bone transplantation group of the comparative example, 1 out of 5 animals underwent bone fusion, and the bone fusion rate was 20%. In addition, when bone fusion was evaluated by CT images after 6 weeks of bone transplantation, as shown in FIG. Figure 7 As shown, in the bone transplantation alone group, gaps were present between bones and sufficient bone healing did not occur, but it was confirmed that sufficient bone healing occurred in the nonwoven fabric combined use group.

[0068] When bone fusion was evaluated by immunostaining images 6 weeks after bone transplantation, it was confirmed that human MSCs were produced in rats in the PCL nonwoven fabric combined group. Specifically, cells that were positive for both HNA (Human Nuclear Antigen) and DAPI were human mesenchymal stem cells, and cells that were positive for only DAPI were rat cells. Figure 8 As shown in the figure, when the HNA image on the left is overlapped with the DAPI image in the middle, the image on the right is obtained. When observing this image, there are a large number of cells that are positive for both HNA and DAPI. Therefore, it can be confirmed that bone fusion is progressing even at the cellular level in the group using the PCL nonwoven fabric.

[0069] This application claims the benefit of priority of Japanese Patent Application No. 2022-100731 filed on June 22, 2022 and Japanese Patent Application No. 2023-54932 filed on March 30, 2023, and includes the specification, claims, drawings, and abstract thereof. The contents disclosed in the above Japanese patent applications are incorporated herein by reference in their entirety.

[0070] Industrial availability

[0071] The tissue regeneration promoting sheet of the present invention is useful because it can stably retain stem cells.

[0072] Explanation of symbols

[0073] 10: Tissue regeneration promoting sheet

[0074] 12: Cell holding sheet

[0075] 12A: First cell holding sheet

[0076] 12B: Second cell holding sheet

[0077] 14: Strengthening sheet

[0078] 20: Support frame

Claims

1. A tissue regeneration promoting sheet comprising a nonwoven fabric, The nonwoven fabric contains fibers containing polycaprolactone as a main component and is capable of permeating a cell suspension containing stem cells.

2. The tissue regeneration promoting sheet according to claim 1, wherein The fiber diameter of the fibers constituting the nonwoven fabric is in the range of 300 nm to 2 μm.

3. The tissue regeneration promoting sheet according to claim 1 or 2, wherein: The nonwoven fabric has a unit area weight of 1 g / m 2 ~3g / m 2 within the range.

4. The tissue regeneration promoting sheet according to any one of claims 1 to 3, wherein The nonwoven fabric has a thickness of 300 μm or more.

5. The tissue regeneration promoting sheet according to any one of claims 1 to 4, wherein The tissue regeneration promoting sheet further comprises a reinforcing sheet, The reinforcing sheet is stacked on the non-woven fabric and can allow the cell suspension to permeate.

6. The tissue regeneration promoting sheet according to claim 5, wherein: The reinforcing sheet is another nonwoven fabric composed of fibers thicker than the fibers constituting the nonwoven fabric.

7. The tissue regeneration promoting sheet according to any one of claims 1 to 6, wherein The nonwoven fabric comprises a first nonwoven fabric and a second nonwoven fabric. The second non-woven fabric is stacked on the first non-woven fabric and is less likely to deform than the first non-woven fabric.

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